Analysis of the low molecular weight peptides of
selected snake venoms
Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften an der Fakultät für Mathematik, Informatik und Naturwissenschaften
der Universität Hamburg
vorgelegt von
Aisha Munawar (M. Phil)
aus Lahore, Pakistan
Die vorliegende Arbeit wurde im Zeitraum von Juli 2009 bis September 2012 in der Arbeitsgruppe von Prof. Betzel am Institut für Biochemie und Molekularbiologie am Fachbereich Chemie der Universität Hamburg angefertigt.
Gutachter:
Herr Prof. C. Betzel Herr Prof. A. Torda
Abstract
I Abstract
Snake venom peptidomes are valuable sources of pharmacologically active compounds. The peptidic fractions (peptides with molecular masses up to 10,000 Da) of the venoms of Vipera
ammodytes meridionalis (Viperinae), the most toxic snake in Europe, Bothrops jararacussu
(Crotalinae), an extremely poisonous snake in South America, Naja mossambica mossambica (Elapinae), a snake from Africa and Notechis ater niger (Acanthophiinae), distributed on the south coast of Australia having a very lethal venom, were analyzed. Liquid chromatography coupled to mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of the four snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences were determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. 3 cytotoxins, 5 BBPs and one bradykinin inhibitor peptide were found in the Elapinae venom. Two neurotoxins, two Kunitz/BPTI type inhibitors and one natriuretic peptide were identified in the Acanthophiinae venom. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far shows a subfamily specificity of the venom peptidome in the Viperidae and Elapidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae and Acanthophiinae venom, in addition to BPP or natriuretic peptides, can contain peptides of the bovine pancreatic trypsin inhibitor family. The venoms of Elapidae family contain three finger toxins which have not been found in the Viperidae family and the absence of three finger toxin type of peptides in the Viperidae venom can mark the point of difference between the Elapidae and Viperidae family. Among the Elapidae family variations were also observed at the subfamily level. The Elapinae family contains cytotoxin type of three finger toxin, while that of Acanthophiinae venom lacks cytotxic peptides and instead contains neurotoxin type of three finger toxins. The Acanthophiinae family contains Kunitz/BPTI inhibitors which were not observed in the Elapinae venom. The MALDI-TOF mass spectrometry provided information for the post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (SQGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom
Abstract
II
natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The Kunitz/BPTI type inhibitors isolated from the venoms of Viperinae and Acanthophiinae subfamily are also homologous to each other, but show variation of the reactive bond residues even within the same venom, suggesting that nature has engineered these peptides to perform a variety of functions by incorporating subtle mutations at convex and exposed binding loop. The results demonstrate that the venoms are rich sources of peptides influencing important physiological systems such as blood pressure regulation, hemostasis, and nervous system. The molecular models of the catalytic complexes of BPP-human ACE and Kunitz/BPTI-serine protease provide insights into the probable binding modes and interactions at the protein-ligand interface. The data can support pharmacological and medical applications.
Zusammenfassung
III Zusammenfassung
Die Peptidome von Schlangengifte sind wertvolle Quellen von pharmakologisch wirksamen Verbindungen. In der vorliegenden Arbeit wurden die peptidischen Fraktionen (Peptide mit einem Molekulargewicht von weniger als von 10,000 Da) der Gifte von Vipera ammodytes
meridionalis (Viperinae), der giftigsten Schlange Europas, Bothrops jararacussu (Crotalinae),
einer extrem giftigen Schlange aus Südamerika, Naja mossambica mossambica (Elapinae), der gefährlichsten Schlange Afrikas und Notechis ater niger (Acanthophiinae), von der Südküste Australiens mit einem sehr tödlichen Gift, analysiert. Flüssigkeits-Chromatographie gekoppelt mit Massenspektrometrie (LC/MS), Elektrosprayionisations-Massenspektrometrie per direkter Probeninfusion (ESI-MS) und Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight-Massenspektrometrie (MALDI-TOF-MS) wurden angewandt, um die Peptide der vier Schlangengifte zu charakterisieren. Im Gift der Crotalinae wurden 32 Bradykinin-potenzierende Peptide (BPPs) identifiziert und ihre Sequenzen wurden bestimmt. Im Peptidom des Viperinae-Gifts wurden 3 Metalloproteinase-Inhibitoren, 10 BPPs und ein Kunitz-Typ-Inhibitor beobachtet. Drei zytotoxische Komponenten, 5 BBPs und ein Bradykinin-Inhibitor-Peptid wurden im Elapinae-Gift gefunden. Zwei Neurotoxine, zwei Kunitz-/BPTI-Typ-Hemmstoffe und ein natriuretisches Peptid wurden im Acanthophiinae-Gift identifiziert. Eine Variabilität am C-Terminus von homologen BPPs, die die pharmakologischen Wirkungen beeinflussen kann, wurde beobachtet. Die erhaltenen Daten zeigen eine Spezifität der Gifte in den Unterfamilien im Peptidom der Viperidae- und Elapidaefamilie: BPPs sind die wichtigsten Peptidkomponenten des Crotalinae-Gift-Peptidoms. In diesem fehlen Kunitz-Typ Inhibitoren (mit einer Ausnahme), während das Gift von Viperinae und Acanthophiinae, zusätzlich zu BPPs oder natriuretischen Peptiden, Peptide der Bovin-Trypsin-Inhibitor-Familie enthalten können. Die Gifte der Elapidae-Familie enthalten sogenannte Dreifinger-Toxine (three finger toxins), die nicht in der Viperidae-Familie gefunden wurden. Das Fehlen von Dreifinger-Giftstoffen im Gift von Viperidae ist der entscheidende Unterschied zwischen dem Gift der Elapidae- und Viperidae-Familien.
Innerhalb der Elapidae-Familie wurden auch auf der Subfamilien-Ebene Unterschiede in der Zusammensetzung des Giftes beobachtet. Das Gift der Elapinae-Familie enthält Cytotoxin vom Typ des Dreifinger-Toxins, dies fehlt im Gift der Acanthophiinae welches stattdessen Neurotoxine vom Typ der Dreifinger-Toxine enthält. Die Acanthophiinae-Familie enthält Kunitz-/BPTI-Inhibitoren, die nicht im Gift der Elapinae-Familie gefunden werden konnten. Die
Zusammenfassung
IV
MALDI-TOF-Massenspektrometrie ergab einen Anhaltspunkt für die post-translationale Phosphorylierung von Serin im BPP aus dem Gift von Bothrops jararacussu (SQGLPPGPPIP). Dies könnte ein Regulationsmechanismus in ihren Interaktionen mit ACE sein und die blutdrucksenkende Wirkung beeinflussen. Die Homologie zwischen BPPs von Viperidae-Schlangen und den Vorstufen des natriuretischen Peptids im Gift von Elapidae-Viperidae-Schlangen verweist auf eine strukturelle Ähnlichkeit zwischen den jeweiligen Peptiden beider Schlangenfamilien. Die Kunitz-/BPTI-Typ-Inhibitoren, die aus den Giften der Viperinae und Acanthophiinae-Unterfamilie isoliert wurden, sind auch homolog zueinander, zeigen aber Unterschiede bei ihren reaktiven Bindungsstellen, selbst innerhalb desselben Giftes, was darauf hindeutet, dass durch Einbau von kleinen Mutationen an konvex und freiliegenden bindenden Loops diese Peptide verschiedene Funktionen haben. Die Ergebnisse zeigen, dass die Gifte von vier Schlangen reiche Quellen für Peptide sind, die wichtige physiologische Systeme wie Regulierung des Blutdrucks, Hämostase und das Nervensystem beeinflussen. Die molekularen Modelle der katalytischen Komplexe von BPP aus Schlangengiften mit menschlichem tACE und der Kunitz-/BPTI in Komplex mit Serinproteasen gibt Einblicke in die wahrscheinlichen Bindungsmodi und Wechselwirkungen an der Protein-Ligand-Schnittstelle. Die Daten können für pharmakologische und medizinische Anwendungen verwendet werden.
Table of Contents V Table of Contents Abstract ... I Zusammenfassung... III List of Abbreviations ... IX
Symbols for Amino Acids ... xi
Introduction ... 1
1.1: Snake venom ... 1
1.2: Chemical arsenal of the snake: nature’s bio resource for drug design ... 5
1.3: Evolution of the snake venom proteins... 7
1.4: Snake venoms analyzed in terms of the thesis ... 10
1.4.1: Bothrops jararacussu ... 10
1.4.2: Vipera ammodytes meridionalis ... 11
1.4.3: Naja mossambica mossambica ... 11
1.4.4: Notechis ater niger... 12
2: Aims and significance of the project ... 13
3: Materials and methods ... 14
3.1: Collection of snake venom ... 14
3.2: Liquid chromatography of crude venom and peptide fractions of Vipera ammodytes meridionalis ... 14
3.3: Liquid chromatography of crude venom and peptide fractions of Bothrops jararacussu 15 3.4: Liquid chromatography of crude venom and peptide fractions of Naja mossambica mossambica ... 15
3.5: Liquid chromatography of crude venom and peptide fractions of Notechis ater niger ... 16
3.6: Liquid chromatography of crude venom of Agkistrodon bilineatus ... 16
3.7: SDS-polyacrylamide gel electrophoresis (PAGE) ... 16
Table of Contents
VI
3.8.1: Angiotensin I-converting enzyme (ACE) assay ... 17
3.8.2: Subtilisin (StmPr1) assay ... 18 3.8.3: Thrombin assay ... 18 3.8.4: Trypsin assay ... 18 3.8.5: Chymotrypsin assay ... 19 3.8.6: Factor Xa assay ... 19 3.8.7: Plasmin assay ... 19
3.8.8: Plasma kallikrein assay ... 20
3.8.9: 20S Proteasome assay ... 20
3.8.10: Snake venom metalloproteinase and serine proteinase assay ... 20
3.2.9: Tryptic digestion and mass spectrometric identification of larger peptides ... 21
3.2.10: Matrix-assisted desorption/ionization time-of-flight mass spectrometry ... 22
3.2.11: Electrospray ionization time-of-flight mass spectrometry... 22
3.2.12: ESI-QTOF mass spectrometry for peptide sequencing ... 23
3.2.13: ESI-FTICR mass spectrometry for peptide sequencing ... 23
3.2.16: Crystallization experiment: soaking of a native StmPr1 crystal with a peptidic fraction from Agkistrodon bilineatus venom... 24
3.2.17: Molecular modelling ... 24
4: Results and Discussion ... 26
4.1: Fractionation of the Vipera ammodytes meridionalis venom by size exclusion chromatography and purification of peptides by liquid chromatography ... 27
4.2: Kunitz-type, ACE and metalloproteinase inhibitors in the Vipera ammodytes meridionalis venom ... 39
4.3: Fractionation of the Bothrops jararacussu venom by size exclusion chromatography and purification of peptides by liquid chromatography ... 43
Table of Contents
VII
4.5: Fractionation of the Naja mossambica mossambica venom by size exclusion
chromatography and purification of peptides by liquid chromatography ... 53
4.6: Cytotoxins, ACE inhibitors and a bradykinin inhibitor peptide in the Naja mossambica mossambica venom ... 62
4.7: Fractionation of the Notechis ater niger venom by size exclusion chromatography and purification of selected peptides by liquid chromatography ... 65
4.8: Neurotoxin, natriuretic and Kunitz type peptides in Notechis ater niger venom ... 70
4.9: Fractionation of the Agkistrodon bilineatus venom by size exclusion chromatography. .. 74
4.9.1: Preparation of crystal complex of a new peptidic inhibitor from Agkistrodon bilineatus venom with bacterial subtilisin Stmpr1... 75
4.9.2: Structural analysis of the crystal complex ... 75
4.10: Molecular modelling of snake venom Kunitz/BPTI inhibitors with trypsin and kallikrein ... 78
4.10.1: Molecular docking of tigerin-1 with trypsin ... 80
4.10.2: Molecular docking of tigerin-1 with the catalytic domain of human plasma kallikrein (pkal 1). ... 81
4.10.3: Molecular docking of tigerin-3 with trypsin ... 83
4.10.4: Molecular docking of protease inhibitor 1with trypsin ... 85
4.11: Molecular modelling of cytotxin-1 with chymotrypsin ... 86
4.12: Molecular modelling and docking of selected BPPs with the catalytic C-domain of human ACE ... 87
5: General discussion ... 104
Conclusion ... 108
Future work ... 109
References ... 110
List of chemicals and GHS hazards ... 127
Table of Contents
VIII
List of Abbreviations
IX List of Abbreviations
A Absorbance
l Length of Spectrophotometer Cell
C Concentration
ACE Angiotensin Converting Enzyme
AChR Acetylcholine receptor
ACN Acetonitrile
BPP Bradykinin potentiating Peptide ANP A type natriuretic peptide BNP B type natriuretic peptide CNP C type natriuretic peptide CID Collision Induced Dissociation CRISP Cysteine-rich secretory proteins
2DE 2 Dimensional Eleophoresis
Ɛ Extinction coefficient
λex Excitation Wavelength
λem Emission Wavelength
ESI Electrospray Ionization
3FTx Three Finger Toxin
FTC Fluorescein thiocarbamoyl-casein FITC Fluorescein isothiocyanate
FTICR Fourier Transform Ion Cyclotron Resonance FPLC Fast Protein Liquid Chromatography
GBL Galactose binding Lectins
Hex Hexoe
HexNAc N-acetylhexoseamine
HPLC High Performance Liquid Chromatography
List of Abbreviations
X
HCl Hydrochloric acid
MALDI-TOF Matrix Assisted Laser Desorption Ionization- Time of Flight
MS Mass spectrometry
M Protein marker
NCBI National Center for Biotechnology Information
NeuAc N-acetylneuraminic
NMR Nuclear Magnetic Resonance
NP Natriuretic Peptide
PAGE Polyacrylamide Gel Electrophoresis PLA2 Phospholipase A2
QTOF Quadruple Time of Flight
RPC Reverse Phase Chromatography
SDS Sodium Dodecyl Sulphate
SEC Size Exclusion Chromatography
SVMP Snake Venom Metalloproteinase
SVSP Snake Venom Serine Proteinase
StmPr1 Stenotrophomonas maltophilia Protease 1
T Temperature
Tris Tris (hydroxymethyl) aminomethane VEGFs Vascular endothelial growth factors NEGFs Nerve endothelial growth factors
η Viscosity
λ Wavelength
Symbols for Amino Acids
xi Symbols for Amino Acids
A Ala Alanine R Arg Arginine N Asn Asparagine D Asp Aspartate C Cys Cysteine E Glu Glutamate Q Gln Glutamine G Gly Glycine H His Histidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine Z Pyr Pyroglutamate
Introduction
1 Introduction
1.1: Snake venom
Snake venom is an exquisitely complex mixture of hundreds of compounds, as proteins, peptides, carbohydrates, nucleosides, amines, free amino acids and lipids. Protein and peptide components comprise 90–95% of the dry weight of the venom [1, 2]. Snakes use their venoms as an offensive weapon in incapacitating and immobilizing their prey, as a defensive tool against their predators, and to aid in digestion, and hence use it as a tool to survive in their particular environment. Venomous snakes in the family Viperidae and Elapidae have a complex mixture of distinct toxic proteins produced in the specialized venom glands located in the upper jaw, which they inject into the prey using fang [3].
Scientists all over the world, study snake venoms and toxins by focusing one or more of the following objectives, i) to determine the mode and mechanism of action of the toxins, ii) to develop antivenoms/antidotes to neutralize the adverse effects of snake venom envenomation, iii) to understand the physiological processes both at cellular and molecular level and to design novel medicines, iv) to develop archetypes of pharmacological agents based on the structure of the toxins, and v) to understand the ecological niche and evolutionary relationship of the snakes [4, 5].
By now a large number of snake venom proteins have been purified and characterized. Some of them exhibit enzymatic activities, whereas others are non enzymatic proteins and polypeptides. Based on their structures, they can be grouped into families (summarized in Fig. 1) [6]. The members of a single family show remarkable similarities in their primary, secondary and tertiary structures, but they often exhibit distinct pharmacological effects [7].
Venom proteins are subjected to accelerated Darwinian evolution [8], and variability of venom composition at the genus, species, subspecies, population and individual levels may endow snakes with the capability to adapt to different ecological niches [9].
In 1994 Marc Wilkins developed the concept of proteome and coined the term [10]. In 1997 he co-wrote and co-edited the first book on proteomics [11]. Proteomics analysis of snake venoms, also known as “snake venomics”, is greatly expanding the knowledge and understanding of these complex secretions, vital to snakes but potentially fatal to humans. The advancement of
Introduction
2
proteomic tools, during the recent years, have accelerated the work in unraveling the venom composition, and therefore paving the way for a deeper understanding of their biological, functional and clinical implications [12]and references therein.
Fig. 1: Composition of the snake venom [6].
Snake venom consists of a wide range of proteins, with a complex proteome. Therefore it is not possible to visualize every component of a proteome using a single proteomic technique. Recent publications in the field of venomics have emphasized the need for multifaceted approaches to
Introduction
3
maximize the protein coverage [6, 13]. Recently, the combinatorial peptide library approach (commercialized as Proteominer TM) has emerged as a powerful tool for mining below the tip of the iceberg, and complements the data gained using the snake venomics protocol towards a complete visualization of the venom proteome [14]. A general scheme of the steps to be followed in a snake venomic analysis is shown in Fig. 2.
Fig. 2: Scheme of the steps typically performed in a snake venomics analyis. CID: Collision induced dissociation; RP-HPLC: Reverse phase HPLC [14].
The proteomic approach has given rise to a comprehensive understanding of the venom complexity, composition, and relative abundance of different protein families, and provides insights to investigators to focus on different issues and identification of novel proteins [15-26]. Studies have shown that the chemical composition of the venoms exhibit geographical variations and may be due to evolutionary environmental pressure acting on isolated populations [27]. Calvete et al. characterized the venom proteome of Bothrops atrox from different geographical
Introduction
4
regions and vindicated the use of a venom signature as a tool to investigate the phylogeography of Bothrops atrox [28]. The snake venom composition is under genetic control and therefore proteome studies could serve as a tool to provide molecular markers for taxonomical purposes [12, 29-31]. However, besides varying between species, venom composition also differs within a species depending on age, season and temperature [27]. Fig. 3 illustrates variation of the venom composition between different species [5].
Fig. 3: Summary of the relative amounts of toxin families in the venoms of; (A) C. d. terrificus venom; (B) C. d. collineatus
venom [5].
There is evidence that individual venom composition can vary through time likely due to the effects of gene regulation, a number of snakes show age related changes in venom composition [9]and references therein. This pattern is interpreted as reflecting ontogenetic changes in gene expression possibly related to diet differences between juvenile (eg. ectothermic prey such as frogs and lizards) and adults (e.g. endothermic prey, such as mammals), tuning the venom toxicity to deal with larger prey by adult snakes [32-34].
Characterizing the large molecular variability within all the major toxin families may contribute to a deeper understanding of the biological effects of the venoms, and poses exciting challenges for delineating structure-function correlations and for designing antivenom production strategies. Snake bite is still a serious threat in both developed and developing countries. Snake envenomation accidents represent a socio-medical problem of considerable magnitude with
Introduction
5
about 2.5 million people bitten by snakes annually around the world, of which more than 100,000 lose their lives. The only effective treatment for systemic envenomation is intravenous administration of antivenom. However many of the antivenom have not achieved optimal protective effects. This is in part due to the fact that antiserum includes numerous antibodies with specificities not confined to the toxic target molecules [35], and also to the fact that the venom used as antigen for serum production may contain poorly immunogenic components, unable to induce production of protective antibodies. The WHO has only recently recognized snake bite as a “neglected tropical disease” [36, 37]. Therefore the knowledge of toxin composition of the venom is of great medical and biotechnological significance, to develop safe and more specific antivenoms, and studies are being carried out to use pooled venoms as a substrate for antivenom production, for designing novel polyvalent pan-generic antivenoms [38-50].
1.2: Chemical arsenal of the snake: nature’s bio resource for drug design
Nature has been the traditional source and inspiration for drug discovery for thousands of years, among which snake venoms form a rich source of bioactive molecules [51].
Snakes have been used in Ayurevedic medicine since the seventh century B.C. to prolong life and treat arthritis and gastrointestinal ailments [52]. Cobra venom has been used since the 1930s to treat conditions as diverse as asthma, polio, multiple sclerosis, rheumatism, severe pain and trigeminal neuralgia [53].
Most venoms are delivered to their prey and consequently the venom peptides and proteins must be stable enough to reach their site of action before being degraded or excreted. This need has resulted in the recruitment of highly stable molecular scaffolds that are resistant to degradation by proteases [54, 55]. The stability is usually attained by disulfide bridges [56], and post-translational modification [57, 58].
Venom components can be used directly or as prototypes of drugs for the treatment of diseases which do not respond to currently available therapies [6, 59]. Some of these compounds have already found preclinical or clinical application for the treatment of hypertension, cardiovascular diseases, multiple sclerosis, diabetes and pain [57]. A well known example is the use of bradykinin-potentiating peptides (BPP), isolated from the Bothrops jararaca venom, which served as an antetype for the first orally-active inhibitor of the angiotensin-converting enzyme (ACE), named captopril [60-62]. BPPs, natriuretic peptides (NPs) and sarafotoxins (SRTXs)
Introduction
6
exert profound effects on the cardiovascular system [63]. Snake venom NPs resemble their mammalian counterparts including atrial natriuretic peptides (ANPs), brain natriuretic peptides (BNPs) and C-type natriuretic peptides (CNPs). Mammalian NPs play a crucial role in natriuresis, diuresis and vasorelaxation [64]. A 38-residues peptide (DNP) was isolated from the
Dendroaspis angusticeps (Green mamba) venom [65], which has been shown to possess
vasodilator, natriuretic and diuretic properties, similar to those of the mammalian NPs [66] and
references therein
. A synthetic analogue of DNP is today a potent therapeutic agent for the treatment of acutely decompensated congestive heart failure [67]. Snake venom sarafotoxins and mammalian endothelins (ETs) are structurally and pharmacologically related peptides exhibiting a potent vasoconstrictor action. They act on the vascular system via identical receptors [68]. Endothelins are very potent vasoconstrictor substances [69]. Disintegrins, found in the venoms of Viperinae and Crotalinae snakes, are non-enzymatic peptides which selectively block integrin receptors. These receptors are located on the cell surface and mediate cell-cell and cell-matrix interactions [70, 71]. Disintegrins and their analogues have the potential to be used as pharmacological tools for the treatment of heart attacks, cancer, osteoporosis and diabetes [72]. A potent peptide antibiotic, cathelicidin-BF, was purified from the venom of Bungarus fasciatus [73]. Further research work based on structure activity relationship, is being carried out, to design novel and cost effective antimicrobial peptides with reduced haemolytic activity [74]. Textilinin-1, which is a 7 kDa Kunitz type serine protease inhibitor isolated from the venom of P. textilis, has been found to be a potent and selective inhibitor of plasmin. In vitro and in vivo studies have shown that this molecule is equally effective and has a better safety profile as compared to aprotinin, therefore it has been suggested as a lead candidate for the replacement of aprotinin as an anti-fibrinolytic agent [75].
The pharmaceutical industry has recognized the enormous potential inherent to venom peptides and has begun to exploit the selectivity and sensitivity fine tuned by evolution [76]. There are approximately 60 peptide drugs on the market with combined sales in 2010 of $ 13 billion [77]. Presently peptides share about 2% of the drugs in the market, and account for 50 % of the drugs in the pipelines of the drug manufacturers [78]. The drugs derived from snake venom, which have been approved or in clinical or preclinical trials are shown in table 1.
Introduction
7
Table 1: Drugs derived from snake venom [78]
1.3: Evolution of the snake venom proteins
The advanced snakes (superfamily Colubroidea) constitute over 80% of the approximately 2,900 species of snake currently described and contain all the known venomous forms. Only about 20 % of the advanced snakes (Atractaspididae, Elapidae, Hydrophidae and Viperidae) have front-fanged delivery systems, and are typically regarded as of major medical interest [79]. The evolutionary studies of the venom are based on the variable nature of the venom. Venoms represent the critical innovation in ophidian evolution that allowed the advanced snakes to transition from a mechanical (constriction) to a chemical (venom) means of subduing and digesting prey larger than themselves [59, 80]. A great deal of work in mining the origin, evolution and phylogeny of the snake venom toxins was done by Fry and colleagues [81-87]. One of the important conclusions was that, the snake venom toxins evolved from recruitment events by which a body protein is recruited into the chemical arsenal of the snake. The toxins often undergo significant variations in sequence and structure, yet typically retain the molecular
Introduction
8
scaffold of the ancestral protein [82]. It is believed that the original PLA2, for example, had a
role in digestion and that upon recruitment of this protein in the venom gland, the ancestral digestive enzyme evolved and through subtle mutations became toxic [84]. Studies on the origin and evolution of the snake venom proteome revealed that CRISPs and kallikrein toxins result from modification of salivary proteins, and that the toxin types, where the ancestral protein was extensively cysteine cross-linked, were the ones that flourished into functionally diverse, novel toxin multigene families [84].
The venomous snake families (Atractaspididae, Elapidae, Viperidae and Colubridae) consist of snakes with venoms that contain shared protein families. Despite of the fact that homologous toxins are found in the venom of these families, the venom of each family has distinct biological activity. The differences in biological characteristics are caused by the variation in amino acid sequence and relative abundance of the related proteins [13]. Viperidae venom toxins can be subdivided into two major groups, enzymatic and non enzymatic toxins. The enzymatic toxins consist of group II PLA2s, serine proteases, metalloproteinases, LAAOs and glutaminyl cyclise.
The non enzymatic toxins include C-type lectins, disintegrins, CNP, CRISPs, VEGFs, NEGFs, cystatin, BPP and Kunitz type protease inhibitors. The Elapidae snakes venom is characterized by a high post and presynaptic neurotoxicity, and contains a wide variety of group 1 PLA2, 3FTx,
CRISPs, Kunitz type protease inhibitors, NGFs, galactose-binding lectins, ANP, cystains and M12B peptidases The Atractaspididae snake venoms have a variety of peptide toxins that affect the cardiovascular system and the Colubridae snake venoms share some similar activities to both the Viperidae and Elapidae snakes [6].
Vipers and elapids are the most distantly related lineages among the Colubroidea, (Fig. 4) [88]. Fry et al. [83], studied eight toxin families, to investigate the origin and recruitment of toxin families into the venom proteome of these snakes. According to them the Kunitz type protease inhibitors, CRISP toxins, GBL toxins, M12B peptidases and NFG toxins were recruited at an early stage before the split of the lineage.
Introduction
9
Fig. 4: Phylogeny of the major families of advanced snakes [88].
Three toxin families, lectin toxins, PLA2, and the natriuretic peptides were recruited at two
independent events. The Elapidae toxins belong to the “pancreatic-type” (group I) PLA2 toxins,
whereas the Viperidae toxins belong to the “synovial-type” (group II) PLA2 toxins. The lectin
protein family was recruited once before the split of the lineage, i.e. the GBL toxins and C-type lectins again in the Viperidae lineage subsequent to its split from the rest of the advance snakes. Hence the vipers contain both the C-type lectins and GBL, whereas all other lineages contain only GBL. On the contrary the actual points of recruitment of the group I PLA2 and natriuretic
toxin families remain unknown. Group I PLA2 toxins have so far only been characterized and
sequenced from elapid venoms. B. J. Fry has stated that the ANP/BNP natriuretic toxins may be another ancient recruitment at the base of the Colubridae tree, where as the CNP natriuretic toxins are an independent recruitment that occurred after the vipers split off from the remainder of the advanced snakes like that of the lectin toxins. The 3FTx family was recruited immediately after the vipers split from the remaining colubroid lineages. A number of other toxin families are presently known only from either elapids or the viperids and may have been recruited into the venom proteome later during the evolution of these lineages. Toxins molecular scaffolds sequenced only from Elapidae venoms include acetylcholinesterase, cobra venom factor, factor Xa prothrombin-activating toxins, factor V toxins, prokinecitin-like peptides, wapins, and toxins containing the SPRY domain. Current viperidae only toxins include myotoxic peptides, S1 peptidases, vascular endothelial growth factor-like toxins, and waglerins. Thus venoms evolved into complex and sophisticated secretions soon after the initial evolution of serous supralabial glands at the base of the colubroid radiation [89]. PII-disintegrins have been found only in the
Introduction
10
viperidae venom, which evolved by the neofunctionalization of disintegrin-like domains of duplicated PIII-SVMP genes. Since PIII-SVMPs exist in all the five families of Colubroidea, it was concluded that disintegrins emerged after the split of Viperidae and Elapidae, but before the separation of Viperidae subfamilies [90].
1.4: Snake venoms analyzed in terms of the thesis
The following four snake venoms were selected from Viperidae and Elapidae snake family (Fig. 5), with different geographical distribution and habitat, to study their peptidic fractions in a comparative way.
Fig. 5: Flow chart displaying the families and geographical distribution of snakes, the venom of which was analyzed in terms of the thesis.
1.4.1: Bothrops jararacussu
This snake is found in South America. It belongs to the subfamily Crotalinae and genus Bothrops. Fig. 5 shows a Bothrops jararacussu snake and its geographical distribution in South America. This snake has an exceptionally large venom output; up to 1000 mg (dry weight) venom can be obtained from a single milking [91]. The venom B. jararacussu is an enormous reservoir of pharmacologically active compounds.
Snake Family Viperidae Bothrops jararacussu (Crotalinae) (Brazil) Vipera a meridionalis (Viperinae) ( Europe) Elapidae Naja mossambica (Elapinae) (Africa)
Notechis ater niger
(Acanthophiinae) (Australia)
Introduction
11
Fig. 6: (A) Bothrops jararacussu snake; (B) Map illustrating the geographical distribution of Bothrops
jararacussu (WHO venomous snake data base).
1.4.2: Vipera ammodytes meridionalis
This snake is from the biodiversity of Europe. It belongs to the subfamily Viperinae and genus Vipera. This snake is of public health significance and the most toxic European snake [92], with an unexplored venom peptidome. It is widely distributed in the eastern part of the continent, Fig. 6.
Fig. 7: (A) V. a. meridionalis; (B) Map illustrating the geographical distribution of the snake (WHO venomous snake data base).
1.4.3: Naja mossambica mossambica
This is a type of spitting cobra native to Africa, Fig. 7. It belongs to the family Elapidae, subfamily Elapinae and genus Naja. It is considered to be one of most dangerous snakes in Africa. Envenoming of the prey by this snake results in severe damage of the local tissue, and venom in the eyes can cause impaired vision or blindness [93].
Introduction
12
Fig. 8: (A) Naja m mossambica; (B) Map illustrating the geographic distribution of the snake (WHO venomous snake data base).
1.4.4: Notechis ater niger
This is an Elapidae snake (subfamily Acanthophiinae, genus Notechis). Snakes of this genus are distributed on the south coast of Australia (Fig. 8). The venom of this snake is highly lethal having neurotoxins, coagulants, haemolysins and myotoxins. Whereas the mainland snakes have a diet including lizards and frogs, the Kangaroo Island snakes of this species prefer to feed on mammals.
Fig. 9: (A) Notechis ater niger; (B) Map illustrating the geographical distribution of the snake (WHO venomous snake data base).
Aims and Significance of the Project
13 2: Aims and significance of the project
The advent of proteome technique has fostered the knowledge gained about the venom proteome composition, protein structure and function. However snake venom peptidomes are a so far mainly neglected/unexplored field and NCBI data base search shows that the snake venom peptide entries are much less as compared to the snake venom proteins. This can be ascribed to the difficulty in obtaining the venom to perform these studies, as the snake venom is very scarce and the peptides are present at a lower concentration as compared to the proteins. Another hypothesis might be that unlike the protein components of the venom, the peptide fractions can not be analyzed by high throughput approaches like 2-D gels followed by automated digestion and MS/MS sequencing. The peptides have to be purified prior to analysis. Purification of the peptides is a difficult task, due to high complexity and the similarity of chemical and physical properties. Also the de novo sequencing of non tryptic peptides is time consuming and difficult. The main objectives of this study were:
Isolation and identification of peptidic inhibitors of pharmacologically interesting enzymes.
Development of analytical methods for the purification and characterization of these peptides.
Molecular modelling and docking to build models of catalytic complexes, of selected peptidic inhibitors with enzymes of interest.
These investigations are of great significance as a better understanding of the snake venom peptides, engineered by nature over millions of years of evolution, would allow the development of new molecules with the potential to pinpoint a specific step of a physiological process such as coagulation, neurotransmission or blood pressure regulation, leading to new drugs with higher specificity. A comparative evaluation of the peptides, present in the snake venoms under study might shed light on the taxonomic and geographical variations in venom among these snakes. The optimization of the analytical methods for the purification and characterization of the peptides would aid in the further investigation of peptides from other snakes venoms.
Materials and Methods
14 3: Materials and methods
3.1: Collection of snake venom
Vipera ammodytes meridionalis venom was collected from snakes originating from the province
Thrace, near the border between Greece and Bulgaria and provided by the Academy of Sciences, Sofia, Bulgaria. Crude venoms from Bothrops jararacussu, Naja mossambica mossambica and
Notechis ater niger were obtained from [Instituto Butantan (São Paulo; Brazil)]. The venoms
were filtered to remove potential mucosal contaminants, lyophilized and stored at -20 °C until required.
3.2: Liquid chromatography of crude venom and peptide fractions of Vipera ammodytes meridionalis
The crude venom was fractionated by size-exclusion chromatography. 50 mg of the venom (dry weight) were dissolved in 0.1 M ammonium acetate buffer, pH 5.0 and applied on a Superdex-75 column, (16 x 60). The chromatography was performed using the same buffer at a flow rate of 1 ml/minute. UV absorbance of the eluate was monitored at 220 and 280 nm. This step was repeated several times to fractionate about 150 mg of the venom. Fractions were collected and subjected to SDS-PAGE on a 15% glycine gel or on 18% Tris/Tricine gel under reducing and non-reducing conditions. The gels were stained with Coomassie Blue. Peptide fractions were further purified by liquid chromatography.
Further separations by HPLC or FPLC were performed on: a) Mono-S Column:
Liquid chromatography of the peptide fraction (Peak 6, Fig. 11) was performed on a Mono-S (5 x 50) cation-exchange column. Peptides were collected with a linear NaCl gradient (0 to1 M), at a flow rate of 1ml/minute, where buffer A was 0.05 M sodium acetate, pH 5 and buffer B was 0.05 M sodium acetate containing 1.0 M NaCl, pH5. b) SOURCE 15RPC Column:
To purify the peptides of the first peak eluting from the Mono-S column, a Source 15 RPC (4.6 x 100) column with a linear gradient of 0–60% consisting of solvent A (0.05% formic acid) and solvent B (0.05% formic acid in acetonitrile, ACN), at a flow rate of 1ml/minute, was used.
Materials and Methods
15 c) PerfectSil 300 ODS-C18 5µm
A C18 (4.6/250 mm) column with a linear gradient system of 0–70% consisting of solvent A (0.05% formic acid in H2O) and solvent B (0.05% formic acid in acetonitrile),
at a flow rate of 1ml/minute, was applied to isolate the peptides from peak 8 (Fig. 10). d) LiChrosorb RP-C8 5µm
A C8 (4.6/150) column with a linear gradient of 0-70% consisting of solvent A (0.05% formic acid in H2O) and solvent B (0.05% formic acid in acetonitrile) was used, at a flow
rate of 1ml/minute, to isolate peptides from peaks 9-11 (Fig. 10).
3.3: Liquid chromatography of crude venom and peptide fractions of Bothrops jararacussu The crude venom was fractionated by size-exclusion chromatography on a Superdex-75 column (10 x 300). A total of 200 mg venom was fractionated using the same buffer and elution conditions as mentioned above, by loading 20 mg (dry weight) of the venom. Peptide fractions were further purified by high pressure liquid chromatography.
a) PLRP Column
Fractions 4-9 obtained from a size exclusion column (Fig. 16), were subjected to further purification on a PLRP column with an asymmetric gradient of 3-60 % consisting of solvent A (20 mM ammonium carbonate) and solvent B (ACN), at a flow rate of 1 ml/ minute.
b) Chromolith C18 (100x4.6) Column
A Chromolith C18 column was used with an asymmetric gradient of 3-40%, consisting of solvent A (0.2% formic acid) and ACN as solvent B, at a flow rate of 2 ml/minute, to further purify the peptides after basic RPC, which showed inhibitory activity towards an angiotensin converting enzyme.
3.4: Liquid chromatography of crude venom and peptide fractions of Naja mossambica mossambica
A total of 200 mg venom was fractionated on Superdex-75 (10 X 300), as mentioned above. Peptide fractions were further separated by FPLC or HPLC.
Materials and Methods
16 a) Resource-S Column
Liquid chromatography of peptide fractions (Peak 5, Fig. 38) was performed on a Resource-S 1ml cation-exchange column. Peptides were separated by a two segment NaCl gradient, 0 to 50% B, 35 CV, and 50-100, in 5 CV, at a flow rate of 1ml/minute, where buffer A was 0.05 M sodium acetate, pH 5.5 and buffer B was 0.05 M sodium acetate containing 1.0 M NaCl, pH 5.5.
b) Vydac C18 (150X4.6) Column
Peptides (Peak 9, Fig. 38) were further separated by RPC, using a C18 column. A linear gradient of 0-75% B was used to elute the fractions, at a flow rate of 0.8 ml/ minute. 0.05% formic acid was used as solvent A and straight acetonitrile was used as solvent B. 3.5: Liquid chromatography of crude venom and peptide fractions of Notechis ater niger A total of 300 mg venom was fractionated on a Superdex-75 (16 x 60) column, under the same conditions as the other venoms. Peptide fractions were further purified by HPLC.
a) SOURCE 5RPC (4.6 x 150) Column
Peak 4 (Fig. 51) was fractionated on a SOURCE 5RPC column, by a linear gradient between 5-75%, at a flow rate of 1ml/minute, for 55 minutes. Solvent A was 0.1% formic acid and solvent B was straight acetonitrile.
b) Chromolith-C18 (100 x 4.6) Column
In order to isolate peptides inhibiting ACE peak 4 (Fig. 51), was filtered through 3 KDa amicon membrane. The peptides present in the filtrate were fractionated on a C18 column, with a linear gradient of 0.3% B to 60 % B, at a flow rate of 1ml/minute, for 40 minutes. 0.2% formic acid was used as solvent A, and straight acetonitrile was used as solvent B.
3.6: Liquid chromatography of crude venom of Agkistrodon bilineatus
The crude venom was fractionated by size-exclusion chromatography, on a Superdex-75 column (16 x 60), under the same conditions as used for other venoms.
3.7: SDS-polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis was performed using 15% glycine or 18% tricine SDS-polyacrylamide, according to the standard protocols [94, 95]. The 18% tricine gel was prepared
Materials and Methods
17
according to the protocol available at
(http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_sm1891.pdf ). The gels were 10 x10 cm and 0.7 mm thick. Before applying the samples to the gels, they were diluted 1:1 with sample buffer and heated to 85 ˚C for 5 minute. The voltage of the chamber was set to 120 V. The protein bands were stained with Coomassie Briliant Blue G250, and de stained with a 20 % acetic acid solution.
3.8: Inhibitory activity of snake venom peptides
The inhibitory activity of snake venom peptides was tested towards the following enzymes, using either chromogenic or fluorogenic substrates. All the assays were downsized to a volume of 100 µl, and the measurements were taken on the TECAN micro plate reader, at room temperature. The inhibitory activity of snake venom peptide was determined by incubating 20 µl of the snake venom fraction with the protease for 15 minutes, and then monitoring the residual protease activity by the addition of the corresponding substrate.
3.8.1: Angiotensin I-converting enzyme (ACE) assay
The ACE activity in the presence of venom peptides was determined by a fluorescence energy transfer assay using Abz-Phe-Arg-Lys (Dnp)-Pro-OH as a substrate [96]. 1 mg of the substrate was weighed and dissolved in 1ml DMSO. The exact concentration of the substrate was determined by taking four different volumes of the substrate stock solution, and constructing a standard curve spectrophometrically at 365 nm,using the molar extinction coefficient of the Dnp group (Ɛ356= 17,300 M-1 cm-1), according to the Beer’s Lambert Law
A= ƐDnp x l x c
The stock solution of the enzyme was prepared by suspending 0.25 UN of ACE in 250 µl of the assay buffer (Dissolve 12.1 g Tris-base, 2.92 g NaCl and 1.36 mg ZnCl2 in 1 liter of deionized
water. Adjust the pH to 7.0 with HCl). Just before the assay an aliquot of the stock solution of ACE was diluted with assay buffer in a ratio of 1:3 (ACE: Buffer). An aliquot of the substrate stock solution was also diluted in a ratio of 1:3 (substrate: buffer). To determine the protease activity 2 µl of the dilute ACE solution was mixed with 96 µl of the buffer, the reaction was started by adding 2 µl of the freshly prepared substrate solution. The fluorescence measurements were made at λex= 320 nm and at λem= 420 nm, for 5 minutes.
Materials and Methods
18 3.8.2: Subtilisin (StmPr1) assay
A bacterial subtilisin (StmPr1) was provided by Dr. Negm [97], to identify and isolate an inhibitor of this protease from snake venom, as a part of an internal collaboration. To determine the serine protease activity the chromophore tetrapeptide Suc-Ala-Ala-Pro-Phe-pNA was used, which is considered to be a non-specific serine protease substrate [98]. After incubation of a serine protease with the substrate nitroaniline is released. This absorbs at 405 nm. The amount of the released nitroaniline is a measure of the activity of the serine protease. A 50 mM stock solution of the substrate was prepared. Before the assay a working solution of the substrate was prepared by diluting 10 µl of the stock solution to 100 µl with the reaction buffer. To measure the protease activity, 10 µl of a diluted protein solution was mixed with 80 µl reaction buffer (20 mM Tris, 20 mM CaCl2, 150 mM NaCl, pH 8), and the reaction was started by addition of 10 the
assay 10µl of the diluted substrate solution. The absorbance was measured at 405 nm for four minutes.
3.8.3: Thrombin assay
The activity of thrombin was determined by using the substrate, Bz-Phe-Val-Arg-pNA [99]. A 20 mM stock solution of substrate was prepared by dissolving 6.82 mg in 500 µl of DMSO. The stock solution of the enzyme was prepared by dissolving 100 UN in 100 µl of the assay buffer (50 mM tris, 100 mM NaCl, pH=8). Just before the assay a working solution of the substrate was prepared by taking 20 µl of the stock solution and diluting to 100 µl with buffer, and that of the enzyme was prepared by diluting in the ratio of 2:5 (enzyme stock solution: buffer). 3µl enzyme was incubated with 84 µl buffer and 13 µl of the substrate working solution were added to start the reaction. Measurements were made by monitoring the absorbance at 405 nm for four minutes.
3.8.4: Trypsin assay
The activity of trypsin was monitored by using the same substrate as thrombin [99]. The assay buffer was also the same as for thrombin. A 0.143 mM stock solution of trypsin was prepared by dissolving 1.8 mg trypsin in 500 µl 1 mM HCl. The substrate stock solution was 20 mM. Prior to the assay a working enzyme solution was prepared by diluting 1.5 µl of the stock solution of the enzyme to 400 µl with the assay buffer. The substrate working solution was prepared by diluting 20 µl of the stock solution to 100 µl with the assay buffer. For the final assay 10 µl of enzyme
Materials and Methods
19
were incubated with 80 µl buffer and 10 µl of substrate were added to start the reaction. Measurements were made by monitoring the absorbance at 405 nm for four minutes.
3.8.5: Chymotrypsin assay
A stock solution of 40 mM of chymoyrypsin was prepared by dissolving 1 mg/ml in 1 mM HCl. The same substrate as that of subtilisin was used for chymotrypsin, and a stock solution of 30 mM was prepared in DMSO. Before starting the assay 2 µl of enzyme stock solution were diluted to 100 µl with buffer (50 mM tris, 10 mM CaCl2, pH 8), and 10 µl of substrate stock
solution were diluted to 100 µl to prepare a working solution. To do the assay 10 µl of enzyme solution was incubated with 70 µl buffer, and 20 µl substrate was added to start the reaction, and change of absorbance was monitored at 405 nm for 4 minutes.
3.8.6: Factor Xa assay
A 50 mM stock solution of the substrate, Z-D-Arg-Gly-Arg-pNA-HCl, was prepared by dissolving 9 mg in 250 µl DMSO [100]. The enzyme was supplied as 13 µl solution with a concentration of 3.8 mg/ml. The enzyme working solution was prepared by diluting 1 µl of the stock enzyme solution to 500 µl with the assay buffer (0.05 M Tris, 5 mM CaCl2, 200 mM NaCl, and pH 8.3). The substrate working solution was prepared by diluting 20 µl stock solution to 100 µl with assay buffer. To do the assay 5 µl of the working solution of the enzyme were incubated with 91 µl assay buffer, and 14 µl of the substrate working solution were added to start the reaction. Change of absorbance was monitored at 405 nm for four minutes.
3.8.7: Plasmin assay
A 50 mM stock solution of the substrate, Bz-Arg-pNA, was prepared by dissolving 10.9 mg in 500 µl DMSO [101]. 150 µg enzyme was dissolved in 100 µl of cold distilled water. The substrate working solution was prepared by diluting 20 µl of the substrate to 100 µl by assay buffer (50 mM Tris-HCl, pH 7.5). The enzyme working solution was prepared by diluting 20 µl of the stock solution to 100 µl with the assay buffer. The assay was done by incubating 10 µl enzyme with 65 µl buffer. The reaction was started by the addition of 25 µl substrate solution, and change of absorbance was monitored at 405 nm for four minutes.
Materials and Methods
20 3.8.8: Plasma kallikrein assay
A solution (10 mM) of the substrate Bz-Pro-Phe-Arg-pNA was prepared by dissolving 1.5 mg in 20 µl DMSO and then diluting it in 500 µl distilled water [102]. A freshly prepared substrate solution was used before the assay. The enzyme was supplied as an aqueous solution (50 µg/96.2 µl). Enzyme working solution was prepared by diluting 8 µl stock solution to 100 µl with the assay buffer (50 mM Tris-HCl, 2mM CaCl2, 150 mM NaCl, pH 7.8). For the assay 10 µl enzyme
were incubated with 70 µl buffer, and 20 µl substrate was added to start the reaction. Change of absorbance was measured at 405 nm for four minutes.
3.8.9: 20S Proteasome assay
560 µg of the substrate Z-Gly-Gly-Leu-AMC, were dissolved in 1ml DMSO, to assay the chymotrypsin like activity of 20S proteasome [103, 104]. 25 µg enzyme was dissolved in 100 µl of assay buffer (20 mM Tris, pH 7.5), to prepare a stock solution. The enzyme working solution was prepared by diluting 7 µl of the stock solution to 100 µl with the assay buffer. For the assay 10 µl of the enzyme working solution were incubated with 85 µl buffer, and 5 µl of substrate were added to start the reaction. The change of fluorescence of the hydrolyzed 7-amido-4-methyl-coumarin (AMC) group was measured at λex= 360 nm and λem= 480 nm, for 10 minutes.
3.8.10: Snake venom metalloproteinase and serine proteinase assay
The caseinolytic activity of SVMP and SVSP was measured by fluorescence substrate, FTC casein [105, 106]. It was prepared as reported previously [107]. 200 mg casein was dissolved in 20 ml buffer (50 mM sodium carbonate, 150 mM NaCl, pH 9.5). After cooling, the mixture was incubated with 8 mg FITC for 8 hours. The reaction mixture was dialyzed, at 4˚C in dark, against 50 mM Tris, pH 8.5, to remove un reacted FITC, for 24 hours and then against 100 mM Tris pH=7.5. 2 ml aliquots of the substrate were prepared and stored at -20˚C.
5µl of FTC-casein were diluted to 100 µl with buffer to prepare a working solution. Reaction mixture containing 5 µl enzyme (1 µg/µl) and 5 µl of the peptide fraction was incubated at room temperature for 15 minutes, followed by the addition of 5 µl of working solution of FTC-casein, 35 µl assay buffer and incubation at 37˚C for 20 minutes. A positive control was prepared by replacing the inhibitor with the assay buffer (50 mM Tris-HCl, pH 7.4), and to prepare a negative control, buffer was added in place of the enzyme and the inhibitor (peptide fraction). Proteolysis was terminated by adding 120 µl 5% TCA and mixing extensively. The reaction mixture was
Materials and Methods
21
allowed to stand for 1/2 h at room temperature, and the TCA-insoluble protein was sediment by centrifugation at 13,000 rpm for 10 minutes. A 90 µl aliquot of the supernatant was diluted with 90 µl of the 0.5 M Tris buffer, pH 8.5, with vigorous mixing to ensure the entire sample was at the assigned pH. Fluorescence was measured at λex = 490 nm and λem at 522 nm. Purified MP-III
60 KDa (B. moojeni), and a serine proteinase (B. alternatus), were used for this assay (kindly provided by Prof. R.K. Arni, UNESP, Brazil)
3.2.9: Tryptic digestion and mass spectrometric identification of larger peptides
Aliquots of the fractions containing peptides of a molecular mass of 6–7 kDa were dried and subsequently dissolved in 6 M urea. To reduce the disulfide bridges, 1.3 μl 100 mM dithiothreitol dissolved in digestion buffer (100 mM NaHCO3, pH 8.3) was added and the
mixture was incubated at 60°C for 10 min. Free cysteine residues were blocked with 1.3 μl iodoacetamide (300 mM dissolved in digestion buffer, incubation for 30 min in the dark). 425 μl digestion buffer and 5μl trypsin solution (sequencing grade modified trypsin; Promega, Madison, USA) at a concentration of 0.25 μg/μl dissolved in re suspension buffer) were added. The mixture was incubated at 37°C for 16 h and afterwards the reaction was stopped by adding formic acid to a final pH of 3.0.
Identification was performed on an Agilent 1100 LC/MSD-trap XCT series system. The electrospray ionization system was the Chip Cube system using a Large capacity Chip (Agilent Technologies, Waldbronn, Germany). Sample loading (5 – 20 μl/sample) onto the enrichment column was performed at a flow rate of 4 μl/min with the mix of the following two mobile phases at a ratio 98:2 (mobile phase A: 0.2% formic acid in H2O; mobile phase B: 100% ACN).
LC gradient was delivered with a flow rate of 400 nl/min. Tryptic peptides were eluted using a linear gradient of 2–40% B in 40 min. For MS experiments, the following mode and tuning parameters were used: scan range: 30–2000 m/z; polarity: positive; capacity voltage: 1900 V; flow and temperature of the drying gas were 4 l/min and 325 °C, respectively. The MS/MS experiments were carried out in auto MS/MS mode using a 4 Da window for precursor ion selection, an absolute threshold of 10,000 after 3 MS/MS spectra. The precursor ion was excluded from fragmentation for one min. The generic files for database searching were generated by Data Analysis software version 3.4; for precursor ion selection a threshold of 5 S/N was applied and the absolute number of compounds was restricted to 1000 per run. Protein
Materials and Methods
22
identification was performed with Mascot online search (www.matrixscience.com) [108]. MS/MS datasets were used to search the spectra against the subset “other lobe-finned fish and tetrapod clade” of the Swiss-Prot database [109]. The experiments were carried out in cooperation with the research group of Prof. H. Schlüter, UKE, Hamburg.
3.2.10: Matrix-assisted desorption/ionization time-of-flight mass spectrometry
MALDI-TOF and MALDI-TOF-TOF analyses were performed on an ultrafleXtreme instrument (Bruker Daltonics, Bremen, Germany). Samples were dried after reversed phase chromatography, dissolved in 30% ACN, 0.1% TFA in H2O and 0.75 μl of the solution was
spotted on a MALDI target plate (MTP AnchorChip 384, Bruker Daltonics). After drying a 0.75 μl MALDI matrix (0.7 mg/ml Cyano-4-hydroxycinnamic acid (Bruker Daltonics) dissolved in 85% ACN, 1 mM NH4H2PO4 and 0.1% TFA dissolved in H2O) were spotted on the sample plate.
Data acquisition was performed in positive ion mode using the flexControl software 3.3. The parameters were set as follows: ion source 1: 25 kV, ion source 2: 23.6 kV, lens: 7.5 kV. MS data were collected automatically using autoXecute. Parameters were set as follows: laser power: 47%; laser shots: 1000; movement, random walk with 100 shots per raster spot. Peaks were selected for LIFT measurement if they met the following criteria: signal to noise > 8, peak intensity > 300.
MS spectra were processed in flexAnalysis (version 3.3, Bruker Daltonics). Further data analysis was performed using BioTools (version3.2, Bruker Daltonics) and Mascot Inhouse Search. Mascot [108] version 2.1.03 was used to search the spectra against the subset “other lobe-finned fish and tetrapod clade”of the Swissprot database. The precursor ion mass tolerance was set to 1 Da, the fragment ion mass tolerance was 0.5 Da.
3.2.11: Electrospray ionization time-of-flight mass spectrometry
ESI analysis was performed on an ESI-TOF-MS system (Agilent Technologies 6224). The samples were dissolved in a 1:1 solution of 0.1% TFA in ACN and 0.1% TFA in water. 1μl of the sample was injected at a flow rate of 0.2ml/min and an internal standard (ESI-TOF reference mix, Agilent Technologies) was used for calibration. Data acquisition was carried out using the Agilent Mass Hunter software (version B.03.01) in positive ESI mode using a gas temperature of 325°C, a gas flow of 10 L/min, a capillary voltage of -4000 V and the fragmentor voltage was set
Materials and Methods
23
to 230 V. Data were acquired in a range of m/z 110 to m/z 3200. These experiments were carried out by the technical staff, department of mass spectrometry, institute of organic chemistry, University of Hamburg.
3.2.12: ESI-QTOF mass spectrometry for peptide sequencing
For protein identification using nano electrospray mass spectrometry, experiments were carried out using an electrospray quadrupole time-of-flight mass spectrometer (Q-TOF-2 electrospray mass spectrometer, Waters, Eschborn, Germany) in the positive ion mode. Raw data were acquired and analyzed using the software MassLynx 4.1 (Micromass, Manchester, United Kingdom). Parameters not specified have been the default parameters of the software. The capillary tip voltage was set to 0.70 kV, the cone voltage to 35 V. For CID experiments, ions were selected within a precursor mass window of ± 1 Da in the quadrupole analyzer and fragmented in the collision cell using Argon as collision gas (Ar) and collision energies of 27 to 35 eV. For peptide identification, peptide tandem mass spectrometry (MS/MS) spectra were deconvoluted by MaxEnt 3 and manually sequenced, supported by PepSeq application for de
novo sequencing (both part of the MassLynx software package). The sequence information of a
few peptides was obtained by using the PEAKS Online software, version 5.2 [110]. These experiments were also carried out at the research group of Prof. H. Schlüter, UKE, Hamburg, by Sönke Harder.
3.2.13: ESI-FTICR mass spectrometry for peptide sequencing
High-resolution mass spectra were acquired using a Finnigan LTQ Fourier transform ion cyclotron resonance (FTICR ULTRA) mass spectrometer (Thermo Fisher, Waltham, USA) equipped with a 7 tesla superconducting magnet. Spectra were acquired at a resolution of 100,000 and the mass error was below 3 ppm at all times. Mass and resolution calibration was performed according to the manufacturer’s recommendations. For CID experiments precursor ions were isolated in the linear ion trap using a mass window of 1.5–2 u and were transferred into the FTICR cell after fragmentation. Collision energies were adjusted in order to detect low intensities of the precursor ion (ca. 20% relative abundance). Electrospray ionization (ESI) of the samples was carried out using a TriVersa Nanomate (Advion BioSystems). An electrospray voltage of 1.5 kV, a pressure of 0.3 psi and a transfer capillary temperature of 200 °C were applied. Samples were diluted in 0.1% formic acid with a 60% methanol part. For data
Materials and Methods
24
processing Qual Browser 2.0.7 (Thermo Fisher) was used and the peptide sequencing was performed manually. These experiments were carried out by Violette Frochaux, Department of Chemistry, Humboldt University, Berlin.
3.2.16: Crystallization experiment: soaking of a native StmPr1 crystal with a peptidic fraction from Agkistrodon bilineatus venom
In an internal collaboration with Dr. Amr Negm, a crystal complex of StmPr1 was prepared with a peptide fraction from the venom of Agkistrodon billineatus, according to the crystallization procedure mentioned in his thesis [97]. The experimental conditions are briefly described here. Native StmPr1 crystals were grown by hanging drop vapour diffusion method, using 1.8 ammonium sulphate and 100 mM Tris-HCl buffer at pH 8.0, by incubating Linbro Plates at 20˚C for three weeks. The crystals were soaked with the peptidic fraction from Agkistrodon bilineatus venom one day before data collection. The data were collected by exposing a single crystal at the synchrotron Consortium-Beamline X13 DESY, Hamburg. The three dimensional model of the complex was built by using the programme Coot [111] and Refmac5 [112]. These experiments were mainly carried out by Dr. Amr Negm.
3.2.17: Molecular modelling
In order to predict the mode of interaction of Kunitz type serine protease inhibitors, isolated in this work, with Trypsin and kallikrein, rigid body docking was performed. Cytotoxin-1, showing inhibitory activity towards chymotrypsin and 20S proteasome, was also docked with chymotrypsin. Structural models of serine protease inhibitor 1, tigerin-1, tigerin-3 and cytotoxin-1 were generated by using the server SWISS-MODEL [cytotoxin-1cytotoxin-13-cytotoxin-1cytotoxin-15].
ClusPro [116-119], a fully automated online server was used to model these complexes. To build a Trypsin-Inhibitor complex, PDB file (PDB code: 3D65) was used. Chymotrypsin-Cytotoxin-1 complex was built by using the PDB file (PDB code: 1MTN). To build the catalytic complex of Kallikrein-Kunitz/BPTI, PDB file (PDB code: 2ANY) was used. In case of each complex generated by the program, the 10 top-ranked complexes from ClusPro were further analyzed, based on the prior knowledge of active site interactions. PDB sum server was used to study the interactions across the protein-protein interface.
Materials and Methods
25
The models of the complexes of the human testis angiotensin converting enzyme and selected bradykinin potentiating peptides, isolated in this work, were built using the flexidock module of the program SYBYL-X, version 1.3. The crystal structure of human tACE in complex with lisinopril (PDB code: 1O86) was used for docking studies. The enzyme was prepared by removing the ligand and water molecules and the energy was minimized. The structures of the bradykinin potentiating peptides, used as ligands, were also prepared by the program SYBYL-X and selecting Gasteiger-Hückel, as the charge model. The ligand was prepositioned in the cavity based on the knowledge obtained from the crystal structure. The biding site was defined around a region of 3 Å and each flexidock simulation was performed with 24,000 generations. The protein-ligand interactions were studied using the PDB sum server.
Results and Discussion
26 4: Results and Discussion
The work flow proposed and followed through this work is presented in Fig. 10. First a SEC separation of the crude venom was performed. The fractions were pooled according to the peaks present in the corresponding chromatogram.
Results and Discussion
27
The pooled fractions containing peptides with molecular masses below 10 kDa were screened for inhibitory activity towards a set of selected enzymes.
The enzymes were chosen so as to give insights into the possible function of the peptides present in the venom. A set of enzymes; thrombin, factor Xa, kallikrein and plasmin, were chosen from the hemostatis system, which might be affected by the injection of the venom into its prey. Trypsin and chymotrypsin were selected, to serve as a probe to identify the serine protease inhibitors, and also to identify Kunitz type inhibitors. Angiotensin converting enzyme, playing a crucial role in the cardiovascular system, was selected to identify the peptides affecting the blood pressure regulatory system of the prey. StmPr1 is a subtilisin like protease, which is produced as an extracellular protease by the bacteria Stenotrophomonas maltophilia, causing disease in humans. This enzyme was included in the work as a part of an internal collaboration in order to identify promising inhibitors of this enzyme within the snake venom, supporting future drug design.
The objective to test the inhibitory activity towards SVSP and SVMP was to look for potential inhibitors that might be responsible for preserving the venom gland from auto digestion, by these enzymes.
The pooled fractions showing inhibitory activity were further purified by liquid chromatography and the active fractions were further characterized by mass spectrometry. The peptides, isolated from the venoms of the four snakes, were classified into protein/peptide families using three indexes/properties: molecular mass, enzyme inhibitory activity and amino acid sequence.
4.1: Fractionation of the Vipera ammodytes meridionalis venom by size exclusion chromatography and purification of peptides by liquid chromatography
The first five fractions contained proteins with molecular masses > 10 000 Da (Fig. 11 B). Fractions 6-11 contained peptides with molecular masses below 10 kDa, which are boxed in Fig. 11A. Fractions 6-12 were screened against the selected enzymes and fractions showing inhibitory activity were subjected to further purification. Peak 6 showed inhibitory activity towards trypsin and peaks 8-11 showed inhibitory activity towards ACE.
